Hybrid pepper &#39;e20s4216&#39;

ABSTRACT

A hybrid pepper designated ‘E20S4216’ is disclosed. The invention relates to the seeds of hybrid pepper ‘E20S4216’ to the plants of hybrid pepper ‘E20S4216’ and to methods for producing a hybrid plant, and to methods for producing other pepper lines, cultivars or hybrids derived from the hybrid pepper ‘E20S4216’.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding. Inparticular, the present invention relates to a new and distinctivepepper (Capsicum annuum) hybrid designated ‘E20S4216’.

BACKGROUND OF THE INVENTION

The bell pepper (Capsicum annuum) originated in Mexico and theneighboring areas of Central America. Soon after Columbus' discovery ofthis plant, it was grown worldwide and used as a spice and a medicine.Today, pepper plants can be found growing wild in tropical areas aroundthe world. Many countries grow it as a crop. Many of the hot peppers canbe found in Latin America and China, but the United States prefers bellpeppers. Peppers are used for fresh consumption, and they are processedinto powders, sauces, and salsas. Many of the new cultivars grown todaycan be traced back to the early plants.

The genus Capsicum and species annuum includes most of the peppers grownin the United States. These can be further grouped into two broadcategories: chile peppers which are pungent (hot) and sweet pepperswhich are non-pungent (mild). The United States produces four percent ofthe world's capsicum peppers (sweet and hot), ranking sixth behindChina, Mexico, Turkey, Spain and Nigeria. Bell peppers are the mostcommon sweet pepper and are found in virtually every retail producedepartment. Grown commercially in most states, the U.S. industry islargely concentrated in California and Florida, which together accountedfor 78% of output in 2000. New Jersey, Georgia, and North Carolina roundout the top five producing states (Economic Research Service, USDA,Vegetables and Melons Outlook/VGS-288/Dec. 14, 2001).

Bell peppers are eaten raw, cooked, immature and mature. Oftennutritional content is altered by the changes in the way they areconsumed. Per capita consumption of bell peppers in 1995 was 6.2 pounds.They are an excellent source of Vitamin C, Vitamin A, and Calcium. Redpeppers have more of these qualities than the immature green peppers.

Peppers grown in temperate regions are herbaceous annuals, but areherbaceous perennials where temperatures do not drop below freezing.Pepper plants' growth habit may be prostrate, compact, or erect, but itis determinate in that after it produces nine to eleven leaves a singlestem terminates in flowers. These plants are grown for the edible fleshyfruit produced by this dichotomous growth. Peppers are non-climactericwhich means they do not produce ethylene. They need to stay on the vineto continue the ripening process. A deep taproot will form if the plantroot system is uninjured during transplanting. The spindle root willdevelop fibrous secondary root systems spreading laterally and downward.On the soil surface the stem will produce adventitious roots, but not aseasily as tomatoes. The leaves of the pepper plant arise singly and aresimple, entire, and asymmetrical. Typical of all Solanaceous plants, theleaves are arranged alternately on the stem. They are shiny and glabrousand vary in shape from broadly ovate to ovate lanceolate. The flowersdevelop singly or in twos or threes continuously as the upper structureof the plant proliferates. The corolla is white and five lobed while theanthers are bluish or yellowish in color. The flowers have an openanther formation and will indefinitely self-pollinate. They are alsopollinated by insects, which increases the chances of cross-pollination.Unlike tomatoes, whose pollen becomes nonviable in high temperatures,the pepper flowers' pollen is not extremely heat sensitive and itremains viable up to 100° Fahrenheit producing fruit throughout theseason.

The fruit of a pepper plant is classified as a berry with colors fromgreen, yellow, red, purple, black, brown, white, and orange. Green is animmature fruit, yet commonly eaten this way, and as the fruit matures itchanges color. In most commercial cultivars color changes are from greento red, green to yellow or green to orange. Usually, fruits of thepurple and white varieties have these colors as they develop, andtherefore do not have a green stage. For fruit to set, the ovaries needto be fertilized. Auxin is then produced by the seeds, which determinefruit cell elongation. The number of seeds fertilized will determine thesize and shape of the fruit. The seed develop on the interior and attachto the veins. Fully developed seed is kidney shaped. There are about4,500 seeds per ounce.

Pepper is an important and valuable field crop. Thus, there is acontinued need for new hybrid peppers.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed toimproved hybrid peppers. In one embodiment, the present invention isdirected to a hybrid pepper, Capsicum annuum, seed designated as‘E20S4216’ having ATCC Accession Number X1. In one embodiment, thepresent invention is directed to a Capsicum annuum pepper plant andparts isolated therefrom produced by growing ‘E20S4216’ pepper seed. Inanother embodiment, the present invention is directed to a Capsicumannuum plant and parts isolated therefrom having all the physiologicaland morphological characteristics of a Capsicum annuum plant produced bygrowing ‘E20S4216’ pepper seed having ATCC Accession Number X1. In stillanother embodiment, the present invention is directed to an F₁ hybridCapsicum annuum pepper seed, plants grown from the seed, and fruitisolated therefrom having ‘E20S4216’ as a parent, where ‘E20S4216’ isgrown from ‘E20S4216’ pepper seed having ATCC Accession Number X1.

Pepper plant parts include pepper leaves, ovules, pollen, seeds, pepperfruits, parts of pepper fruits, flowers, cells, and the like. In anotherembodiment, the present invention is further directed to pepper leaves,ovules, pollen, seeds, pepper fruits, parts of pepper fruits, and/orflowers isolated from ‘E20S4216’ pepper plants. In certain embodiments,the present invention is further directed to pollen or ovules isolatedfrom ‘E20S4216’ pepper plants. In another embodiment, the presentinvention is further directed to protoplasts produced from ‘E20S4216’pepper plants. In another embodiment, the present invention is furtherdirected to tissue culture of ‘E20S4216’ pepper plants, and to pepperplants regenerated from the tissue culture, where the plant has all ofthe morphological and physiological characteristics of ‘E20S4216’pepper. In certain embodiments, tissue culture of ‘E20S4216’ pepperplants is produced from a plant part selected from leaf, anther, pistil,stem, petiole, root, root tip, fruit, seed, flower, cotyledon,hypocotyl, embryo and meristematic cell.

In yet another embodiment, the present invention is further directed toa method of selecting pepper plants, by a) growing ‘E20S4216’ pepperplants where the ‘E20S4216’ plants are grown from pepper seed havingATCC Accession Number X1 and b) selecting a plant from step a). Inanother embodiment, the present invention is further directed to pepperplants, plant parts and seeds produced by the pepper plants where thepepper plants are isolated by the selection method of the invention.

In another embodiment, the present invention is further directed to amethod of making pepper seeds by crossing a pepper plant grown from‘E20S4216’ pepper seed having ATCC Accession Number X1 with anotherpepper plant, and harvesting seed therefrom. In still anotherembodiment, the present invention is further directed to pepper plants,pepper parts from the pepper plants, and seeds produced therefrom wherethe pepper plant is grown from seed produced by the method of makingpepper seed of the invention. In some embodiments, the pepper plantgrown from pepper seed produced by the method of making pepper seed is atransgenic pepper plant.

In another embodiment, the present invention is further directed to amethod of making pepper variety ‘E20S4216’ by selecting seeds from thecross of one ‘E20S4216’ plant with another ‘E20S4216’ plant, a sample of‘E20S4216’ pepper seed having been deposited under ATCC Accession NumberX1.

According to the invention, there is provided a hybrid pepper plantdesignated ‘E20S4216’. This invention thus relates to the seeds ofhybrid pepper ‘E20S4216’, to the plants of pepper ‘E20S4216’ and tomethods for producing a pepper plant produced by crossing hybrid pepper‘E20S4216’ with itself or another pepper plant, and to methods forproducing a pepper plant containing in its genetic material one or moretransgenes and to the transgenic pepper plants produced by that method.This invention also relates to methods for producing other peppercultivars or hybrids derived from hybrid pepper ‘E20S4216’ and to thepepper cultivars and hybrids derived by the use of those methods. Thisinvention further relates to pepper seeds and plants produced bycrossing hybrid pepper ‘E20S4216’ with another pepper cultivar.

In another embodiment, the present invention is directed to methods forproducing a pepper plant containing in its genetic material one or moretransgenes and to the transgenic pepper plant produced by those methods.

In another embodiment, the present invention is directed to single geneconverted plants of hybrid pepper ‘E20S4216’. The single transferredgene may preferably be a dominant or recessive allele. Preferably, thesingle transferred gene will confer such trait as sex determination,herbicide resistance, insect resistance, resistance for bacterial,fungal, or viral disease, improved harvest characteristics, enhancednutritional quality, or improved agronomic quality. The single gene maybe a naturally occurring pepper gene or a transgene introduced throughgenetic engineering techniques.

In another embodiment, the present invention is directed to methods fordeveloping pepper plants in a pepper plant breeding program using plantbreeding techniques including recurrent selection, backcrossing,pedigree breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection and transformation. Markerloci such as restriction fragment polymorphisms or random amplified DNAhave been published for many years and may be used for selection (See,Pierce et al., HortScience (1990) 25:605-615; Wehner, T., CucurbitGenetics Cooperative Report, (1997) 20: 66-88; and Kennard et al.,Theorical Applied Genetics (1994) 89:217-224). Seeds, pepper plants, andparts thereof produced by such breeding methods are also part of theinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The selected germplasm is crossed in order torecombine the desired traits and through selection varieties or parentlines are developed. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm. These important traits may include higher yield, fieldperformance, fruit and agronomic quality such as fruit shape and length,resistance to diseases and insects, and tolerance to drought and heat.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection. Theseprocesses, which lead to the final step of marketing and distribution,usually take from five to ten years from the time the first cross orselection is made.

One goal of pepper plant breeding is to develop new, unique and superiorpepper cultivars. A breeder can initially select and cross two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. Moreover, a breeder can generate multipledifferent genetic combinations by crossing, selfing, and mutations. Aplant breeder can then select which germplasms to advance to the nextgeneration. These germplasms may then be grown under differentgeographical, climatic, and soil conditions, and further selections canbe made during, and at the end of, the growing season.

The development of commercial pepper cultivars thus requires thedevelopment of pepper parental lines, the crossing of these lines, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichlines are developed by selfing and selection of desired phenotypes. Thenew lines are crossed with other lines and the hybrids from thesecrosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding may be used to transfer genes for a simply inherited,highly heritable trait into a desirable homozygous cultivar or line thatis the recurrent parent. The source of the trait to be transferred iscalled the donor parent. The resulting plant is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding may also be used introducing new traits into peppervarieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in Principles of Cultivar Development by Fehr, MacmillanPublishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous lines in a breeding program. Double haploids are producedby the doubling of a set of chromosomes from a heterozygous plant toproduce a completely homozygous individual. For example, see Wan, etal., Theor. Appl. Genet., 77:889-892, 1989.

Additional non-limiting examples of breeding methods that may be usedinclude, without limitation, those found in Principles of PlantBreeding, John Wiley and Son, pp. 115-161, 1960; Allard, 1960; Simmonds,1979; Sneep et al., 1979; Fehr, 1987; “Carrots and Related VegetableUmbelliferae”, Rubatzky, V. E., et al., 1999.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Covered cultivation. Any type of cultivation where the plants are notexposed to direct sunlight. The covering includes but is not limited togreenhouses, glasshouses, net-houses, plastic houses and tunnels.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Fructose content. As used herein, “fructose content” refers to thequantity of fructose in a green pepper fruit in mg/kg of fresh weight.

Glucose content. As used herein, “glucose content” refers to thequantity of glucose in a green pepper fruit in mg/kg of fresh weight.

Green pepper plant. As used herein, a “green pepper plant” is a plantthat is developed for the harvest of green pepper fruits.

Internode. An “internode” refers to the stem segment between nodes.

Pepper fruit. As used herein, a “pepper fruit” is a fruit produced by aCapsicum annuum plant and is commonly referred to as a bell pepper. Thecolor of a pepper fruit can be green, red, yellow, orange and, morerarely, white, black, and brown, depending on when they are harvestedand the specific cultivar. Green peppers are unripe bell peppers, whilethe others are all ripe, with the color variation based on cultivarselection.

Propagate. To “propagate” a plant means to reproduce the plant by meansincluding, but not limited to, seeds, cuttings, divisions, tissueculture, embryo culture or other in vitro method.

Quantitative Trait Loci (QTL). As used herein, “quantitative trait loci”refers to genetic loci that control to some degree numericallyrepresentable traits that are usually continuously distributed.

Regeneration. As used herein, “regeneration” refers to the developmentof a plant from tissue culture.

Single gene converted. As used herein, “single gene converted” or“conversion plant” refers to plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of an inbred arerecovered in addition to the single gene transferred into the inbred viathe backcrossing technique or via genetic engineering.

Transgene. As used herein, a “transgene” is a gene taken or copied fromone organism and inserted into another organism. A transgene may be agene that is foreign to the receiving organism or it may be a modifiedversion of a native, or endogenous, gene.

Overview of the Hybrid Pepper Variety ‘E20S4216’

The taste of pepper fruit can vary with growing conditions andpost-harvest storage treatment. In general, the sweetest pepper fruitare fruit allowed to ripen fully on the plant, while fruit harvestedgreen are less sweet. Green peppers are unripe peppers, and typically,because they are unripe, green peppers are less sweet and slightly morebitter than yellow, orange, brown or red peppers.

Hybrid pepper ‘E20S4216’ produces ripe fruit having a medium orangecolor, and a short, narrowly triangular shape. Fruit of the hybridpepper ‘E20S4216’ matures early and can be grown worldwide.

Additionally, hybrid pepper ‘E20S4216’ has shown uniformity andstability for the traits, within the limits of environmental influencefor the traits. Hybrid pepper ‘E20S4216’ has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in ‘E20S4216’.

Objective Description of Hybrid Pepper ‘E20S4216’

Hybrid pepper variety ‘E20S4216’ has the following morphologic and othercharacteristics:

General:

-   -   Type: Mini conical pepper    -   Usage: Fresh market    -   Type of culture: Open and covered cultivation

Plant:

-   -   Seedling (anthocyanin coloration of hypocotyl): Present    -   Shortened internode: Absent    -   Height: Medium    -   Flower: Present

Fruit:

-   -   Color before maturity: Green    -   Intensity of color before maturity: Medium    -   Length: Short (5 cm)    -   Diameter: Narrow (2.5 cm)    -   Predominant shape of longitudinal section: Narrowly triangular    -   Color at maturity: Orange    -   Intensity of color at maturity: Medium    -   Predominant number of locules: Predominantly two    -   Capsaicin in placenta: Absent    -   Time of maturity: Early

Disease/Pest Resistance:

-   -   Tobamovirus pathotype P₀: Resistant    -   Tobamovirus pathotype P₁: Susceptible    -   Tobamovirus pathotype P₁₋₂: Susceptible    -   Tobamovirus pathotype P₁₋₂₋₃: Susceptible    -   Potato Virus Y pathotype P_(o): Susceptible    -   Potato Virus Y pathotype P₁: Susceptible    -   Potato Virus Y pathotype P₁₋₂: Susceptible    -   Phytophthora capsici: Susceptible    -   Tomato Spotted Wilt Virus (TSWV): Susceptible    -   Cucumber Mosaic Virus (CMV): Susceptible    -   Xanthomonas campestris pv. vesicatoria: Susceptible

Comparisons to Most Similar Variety

Table 1 below compares some of the characteristics of hybrid peppervariety ‘E20S4216’ with the most similar variety, ‘E499531’. Column 1lists the characteristics, column 2 shows the characteristics for mostsimilar pepper variety ‘E499531’, and column 3 shows the characteristicsfor hybrid pepper variety ‘E20S4216’.

TABLE 1 Characteristic ‘E499531’ ‘E20S4216’ Plant length Medium LongerFruit size Small Smaller

Further Embodiments

This invention is also directed to methods for producing a pepper plantby crossing a first parent pepper plant with a second parent pepperplant, wherein the first or second pepper plant is the pepper plant‘E20S4216’. Further, both first and second parent pepper plants may be‘E20S4216’. Therefore, any methods using pepper hybrid ‘E20S4216’ arepart of this invention: selfing, backcrosses, hybrid breeding, andcrosses to populations. Any plants produced using pepper hybrid‘E20S4216’ as at least one parent are within the scope of thisinvention.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

This invention also is directed to methods for producing a pepper plantby crossing a first parent pepper plant with a second parent pepperplant wherein either the first or second parent pepper plant is a hybridpepper plant of hybrid ‘E20S4216’. Further, both first and second parentpepper plants can come from the hybrid pepper ‘E20S4216’. All plantsproduced using hybrid pepper ‘E20S4216’ as a parent are within the scopeof this invention, including plants derived from hybrid pepper‘E20S4216’.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which pepper plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, leaves,stems, and the like.

As it is well known in the art, tissue culture of pepper can be used forthe in vitro regeneration of pepper plants. Tissues cultures of varioustissues of pepper and regeneration of plants therefrom are well knownand published. By way of example, tissue cultures, some comprisingorgans to be used to produce regenerated plants, have been described inBurza, et al., Plant Breeding. 1995, 114: 4, 341-345, Pellinen,Angewandte Botanik. 1997, 71: 3/4, 116-118, Kuijpers, et al., Plant CellTissue and Organ Culture. 1996, 46: 1, 81-83, Colijn-Hooymans, et al.,Plant Cell Tissue and Organ Culture. 1994, 39: 3, 211-217, Lou, et al.,HortScience. 1994, 29: 8, 906-909, Tabei, et al., Breeding Science.1994, 44: 1, 47-51, Sarmanto, et al., Plant Cell Tissue and OrganCulture 31:3 185-193 (1992), Cade, et al., Journal of the AmericanSociety for Horticultural Science 115:4 691-696 (1990), Chee, et al.,HortScience 25:7, 792-793 (1990), Kim, et al., HortScience 24:4 702(1989), Punja, et al., Plant Cell Report 9:2 61-64 (1990). Pepper plantscould be regenerated by somatic embryogenesis. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are “conventional” in the sense that they are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce pepper plants having the physiological and morphologicalcharacteristics of hybrid pepper ‘E20S4216’.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed hybrid.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed pepper plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the pepper plant(s).

Expression Vectors for Pepper Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalswhich confers resistance to kanamycin (Fraley, et al., Proc. Natl. Acad.Sci. U.S.A., 80:4803 (1983)). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin (Vanden Elzen, et al., Plant Mol. Biol.,5:299 (1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant (Hayford, et al., Plant Physiol.86:1216 (1988), Jones, et al., Mol. Gen. Genet., 210:86 (1987), Svab, etal., Plant Mol. Biol. 14:197 (1990), Hille, et al., Plant Mol. Biol.7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai, et al.,Nature 317:741-744 (1985), Gordon-Kamm, et al., Plant Cell 2:603-618(1990) and Stalker, et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet. 13:67 (1987),Shah, et al., Science 233:478 (1986), Charest, et al., Plant Cell Rep.8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include .alpha.-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri, et al.,EMBO J. 8:343 (1989), Koncz, et al., Proc. Natl. Acad. Sci. U.S.A.84:131 (1987), DeBlock, et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available (Molecular Probes publication2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J. Cell Biol.115:151a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie, et al., Science 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Pepper Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inpepper. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in pepper. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See, Ward,et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., Proc. Natl. Acad. Sci. U.S.A.90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey, et al., Mol. Gen.Genetics 227:229-237 (1991) and Gatz, et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, et al., Mol. Gen.Genetics 227:229-237 (1991). A particularly preferred inducible promoteris a promoter that responds to an inducing agent to which plants do notnormally respond. An exemplary inducible promoter is the induciblepromoter from a steroid hormone gene, the transcriptional activity ofwhich is induced by a glucocorticosteroid hormone. Schena, et al., Proc.Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inpepper or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in pepper.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last, etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten, et al., EMBOJ. 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova, et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/Nco1 fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/Nco1 fragment), represents a particularly useful constitutivepromoter. See, PCT Application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin pepper. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in pepper. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science 23:476-482(1983) and Sengupta-Gopalan, et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson, et al., EMBO J. 4(11):2723-2729(1985) and Timko, et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell, et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero, et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell, et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol. 9:3-17 (1987), Lerner, et al., Plant Physiol.91:124-129 (1989), Fontes, et al., Plant Cell 3:483-496 (1991),Matsuoka, et al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould, et al.,J. Cell. Biol. 108:1657 (1989), Creissen, et al., Plant J. 2:129 (1991),Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell 39:499-509 (1984), Steifel, et al., Expression of a maizecell wall hydroxyproline-rich glycoprotein gene in early leaf and rootvascular differentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is pepper. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Methods in Plant Molecular Biology and Biotechnology, Glick andThompson Eds., CRC Press, Inc., Boca Raton, pp. 269-284 (1993). Mapinformation concerning chromosomal location is useful for proprietaryprotection of a subject transgenic plant. If unauthorized propagation isundertaken and crosses made with other germplasm, the map of theintegration region can be compared to similar maps for suspect plants,to determine if the latter have a common parentage with the subjectplant. Map comparisons would involve hybridizations, RFLP, PCR, SSR andsequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfalvum); Martin, et al., Science 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser, et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See, PCT Application US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub, et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani, etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt, etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang, et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See, PCTApplication WO 93/02197 in the name of Scott, et al., which disclosesthe nucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also, Kramer, et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck, et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See, PCT Application WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. See,Taylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb, et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J. 7:1241 (1988), and Miki, et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also, Umaballava-Mobapathie in TransgenicResearch. 1999, 8: 1, 33-44 that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentApplication No. 0 242 246 to Leemans, et al., DeGreef, et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori, et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and International Publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the pepper, for example, by transforming aplant with a soybean ferritin gene as described in Goto, et al., ActaHorticulturae. 2000, 521, 101-109.

B. Increased sweetness of the pepper by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See, Penarrubia, et al., Biotechnology. 1992, 10: 561-564.

4. Genes that Control Male-Sterility:

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac—PPT. See, International Publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See, InternationalPublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See, Paul, et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Pepper Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science 227:1229 (1985), Curtis, et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Tones, et al., Plant CellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant, et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Reports8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, October), 357-359(1992), Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490(1993). Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.;Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford, et al., Part.Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988),Klein, et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., PhysiolPlant 7:206 (1990), Klein, et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes, et al., EMBO J., 4:2731 (1985), Christou,et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.199:161 (1985) and Draper, et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T Biologia Plantarum 40(4): 507-514(1997/98), Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell 4:1495-1505 (1992) and Spencer, et al., Plant Mol. Biol.24:51-61 (1994). See also, Chupean, et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of pepper target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic pepper line. Alternatively, a genetic trait which hasbeen engineered into a particular pepper cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Cregan et al., “An Integrated Genetic Linkage Map of theSoybean Genome” Crop Science 39:1464-1490 (1999), and Berry et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties”Genetics 165:331-342 (2003), each of which are incorporated by referenceherein in their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci forhybrid pepper ‘E20S4216’.

Primers and PCR protocols for assaying these and other markers aredisclosed in the Soybase (sponsored by the USDA Agricultural ResearchService and Iowa State University). In addition to being used foridentification of hybrid pepper ‘E20S4216’ and plant parts and plantcells of variety hybrid pepper ‘E20S4216’, the genetic profile may beused to identify a pepper plant produced through the use of hybridpepper ‘E20S4216’ or to verify a pedigree for progeny plants producedthrough the use of hybrid pepper ‘E20S4216’. The genetic marker profileis also useful in breeding and developing backcross conversions.

The present invention comprises a pepper plant characterized bymolecular and physiological data obtained from the representative sampleof said variety deposited with the American Type Culture Collection(ATCC). Further provided by the invention is a pepper plant formed bythe combination of the disclosed pepper plant or plant cell with anotherpepper plant or cell and comprising the homozygous alleles of thevariety.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. See for example, Gong, L., et al., “Microsatellites for thegenus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepoL.” Theor Appl Genet. (June 2008) 117(1): 37-48. Another advantage ofthis type of marker is that, through use of flanking primers, detectionof SSRs can be achieved, for example, by the polymerase chain reaction(PCR), thereby eliminating the need for labor-intensive Southernhybridization. The PCR detection is done by use of two oligonucleotideprimers flanking the polymorphic segment of repetitive DNA. Repeatedcycles of heat denaturation of the DNA followed by annealing of theprimers to their complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase, comprise themajor part of the methodology. Microsatellites for the genus Cucurbitaand an SSR-based genetic linkage map of Cucurbita pepo.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for pepper plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., 1985), including monocots(see, e.g., Dekeyser et al., 1990; Terada and Shimamoto, 1990); atandemly duplicated version of the CaMV 35S promoter, the enhanced 35Spromoter (P-e35S) the nopaline synthase promoter (An et al., 1988), theoctopine synthase promoter (Fromm et al., 1989); and the figwort mosaicvirus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and anenhanced version of the FMV promoter (P-eFMV) where the promotersequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus19S promoter, a sugarcane bacilliform virus promoter, a commelina yellowmottle virus promoter, and other plant DNA virus promoters known toexpress in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can beused for expression of an operably linked gene in plant cells, includingpromoters regulated by (1) heat (Callis et al., 1988), (2) light (e.g.,pea rbcS-3A promoter, Kuhlemeier et al., 1989; maize rbcS promoter,Schaffner and Sheen, 1991; or chlorophyll a/b-binding protein promoter,Simpson et al., 1985), (3) hormones, such as abscisic acid (Marcotte etal., 1989), (4) wounding (e.g., wunl, Siebertz et al., 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., 1987; Schernthaner et al., 1988; Bustos et al., 1989).

Exemplary nucleic acids which may be introduced to the pepper lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a pepper plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a pepper plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(Bt) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference it their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (i.e., aribozyme) engineered to cleave a desired endogenous mRNA product (seefor example, Gibson and Shillito, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of the present invention.

Single-Gene Conversions

When the terms pepper plant, hybrid, cultivar or pepper line are used inthe context of the present invention, this also includes any single geneconversions. The term “single gene converted plant” as used hereinrefers to those pepper plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a cultivar arerecovered in addition to the single gene transferred into the line viathe backcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into theline. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental pepper plantsfor that line, backcrossing 1, 2, 3, 4, 5, 6, 7, 8, or more times to therecurrent parent. The parental pepper plant which contributes the genefor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental pepper plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a pepperplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, modified fatty acid metabolism, modified carbohydratemetabolism, herbicide resistance, nematode resistance, resistance forbacterial, fungal, or viral disease, insect resistance, enhancednutritional quality, industrial usage, yield stability and yieldenhancement. These genes are generally inherited through the nucleus.Several of these single gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957 and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of pepper andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience. 1992, 27: 9,1030-1032 Teng, et al., HortScience. 1993, 28: 6, 669-1671, Zhang, etal., Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb, etal., Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis, etal., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata,et al., Journal for the American Society for Horticultural Science.2000, 125: 6, 669-672, and Ibrahim, et al., Plant Cell, Tissue and OrganCulture. (1992), 28(2): 139-145. It is clear from the literature thatthe state of the art is such that these methods of obtaining plants areroutinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce pepper plants having the physiological andmorphological characteristics of the hybrid ‘E20S4216’.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a pepper plantby crossing a first parent pepper plant with a second parent pepperplant wherein the first or second parent pepper plant is a pepper plantof hybrid ‘E20S4216’. Further, both first and second parent pepperplants can come from pepper hybrid ‘E20S4216’. Thus, any such methodsusing pepper hybrid ‘E20S4216’ are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using pepper hybrid ‘E20S4216’ as at least oneparent are within the scope of this invention, including those developedfrom cultivars derived from pepper hybrid ‘E20S4216’. Advantageously,this pepper cultivar could be used in crosses with other, different,pepper plants to produce the first generation (F₁) pepper hybrid seedsand plants with superior characteristics. The cultivar of the inventioncan also be used for transformation where exogenous genes are introducedand expressed by the cultivar of the invention. Genetic variants createdeither through traditional breeding methods using pepper hybrid‘E20S4216’ or through transformation of hybrid ‘E20S4216’ by any of anumber of protocols known to those of skill in the art are intended tobe within the scope of this invention.

The following describes breeding methods that may be used with pepperhybrid ‘E20S4216’ in the development of further pepper plants. One suchembodiment is a method for developing progeny pepper plants in a pepperplant breeding program comprising: obtaining the pepper plant, or a partthereof, of hybrid ‘E20S4216’, utilizing said plant or plant part as asource of breeding material, and selecting a pepper hybrid ‘E20S4216’progeny plant with molecular markers in common with hybrid ‘E20S4216’and/or with morphological and/or physiological characteristics selectedfrom the characteristics listed above. Breeding steps that may be usedin the pepper plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers) and the making ofdouble haploids may be utilized.

Another method involves producing a population of pepper hybrid‘E20S4216’ progeny pepper plants, by crossing hybrid ‘E20S4216’ withanother pepper plant, thereby producing a population of pepper plants,which, on average, derive 50% of their alleles from pepper hybrid‘E20S4216’. A plant of this population may be selected and repeatedlyselfed or sibbed with a pepper plant resulting from these successivefilial generations. One embodiment of this invention is the peppercultivar produced by this method and that has obtained at least 50% ofits alleles from pepper hybrid ‘E20S4216’.

Additional methods include, without limitation, chasing selfs. Chasingselfs involves identifying inbred plants among pepper plants that havebeen grown from hybrid pepper seed. Once the seed is planted, the inbredplants may be identified and selected due to their decreased vigorrelative to the hybrid plants that grow from the hybrid seed. Bylocating the inbred plants, isolating them from the rest of the plants,and self-pollinating them (i.e., “chasing selfs”), a breeder can obtainan inbred line that is identical to an inbred parent used to produce thehybrid.

Accordingly, another aspect of the present invention relates a methodfor producing an inbred pepper variety by: planting seed of the peppervariety ‘E20S4216’; growing plants from the seed; identifying one ormore inbred pepper plants; controlling pollination in a manner whichpreserves homozygosity of the one or more inbred plants; and harvestingresultant seed from the one or more inbred plants. The step ofidentifying the one or more inbred pepper plants may further includeidentifying plants with decreased vigor, i.e., plants that appear lessrobust than plants of the pepper variety ‘E20S4216’. Pepper plantscapable of expressing substantially all of the physiological andmorphological characteristics of the parental inbred lines of peppervariety ‘E20S4216’ include pepper plants obtained by chasing selfs fromseed of pepper variety ‘E20S4216’.

One of ordinary skill in the art will recognize that once a breeder hasobtained inbred pepper plants by chasing selfs from seed of peppervariety ‘E20S4216’, the breeder can then produce new inbred plants suchas by sib-pollinating, or by crossing one of the identified inbredpepper plant with a plant of the pepper variety ‘E20S4216’.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes pepperhybrid ‘E20S4216’ progeny pepper plants comprising a combination of atleast two hybrid ‘E20S4216’ traits selected from the group consisting ofthose listed above or the hybrid ‘E20S4216’ combination of traits listedin the Summary of the Invention, so that said progeny pepper plant isnot significantly different for said traits than pepper hybrid‘E20S4216’ as determined at the 5% significance level when grown in thesame environmental conditions. Using techniques described herein,molecular markers may be used to identify said progeny plant as a pepperhybrid ‘E20S4216’ progeny plant. Mean trait values may be used todetermine whether trait differences are significant, and preferably thetraits are measured on plants grown under the same environmentalconditions. Once such a variety is developed its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of pepper hybrid ‘E20S4216’ may also be characterized throughtheir filial relationship with pepper hybrid ‘E20S4216’, as for example,being within a certain number of breeding crosses of pepper hybrid‘E20S4216’. A breeding cross is a cross made to introduce new geneticsinto the progeny, and is distinguished from a cross, such as a self or asib cross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween pepper hybrid ‘E20S4216’ and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4, or 5breeding crosses of pepper hybrid ‘E20S4216’.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which pepper plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as fruit, leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,seeds, stems and the like.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

DEPOSIT INFORMATION

A deposit of the hybrid pepper ‘E20S4216’ is maintained by Enza ZadenUSA, Inc., having an address at 7 Harris Place, Salinas, Calif. 93901,United States. Access to this deposit will be available during thependency of this application to persons determined by the Commissionerof Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14and 35U.S.C. §122. Upon allowance of any claims in this application, allrestrictions on the availability to the public of the variety will beirrevocably removed by affording access to a deposit of at least 2,500seeds of the same variety with the American Type Culture Collection,(ATCC), P.O. Box 1549, MANASSAS, Va. 20108 USA.

Applicants have made available to the public without restriction adeposit of at least 2500 seeds of hybrid pepper ‘E20S4216’ with theAmerican Type Culture Collection (ATCC), P.O. Box 1549, MANASSAS, Va.20108 USA, with a deposit on (Date) which has been assigned ATCC numberX1.

The deposit will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the effective life of the patent, whichever is longer,and will be replaced if a deposit becomes nonviable during that period.

1. Hybrid pepper seed designated as ‘E20S4216’ having ATCC AccessionNumber X1.
 2. A pepper plant produced by growing the seed of claim
 1. 3.A plant part from the plant of claim
 2. 4. The plant part of claim 3,wherein said part is a leaf, a seed, a fruit, a cell, or a portionthereof.
 5. A pepper plant having all the physiological andmorphological characteristics of the pepper plant of claim
 2. 6. A plantpart from the plant of claim
 5. 7. The plant part of claim 6, whereinsaid part is a leaf, a seed, a fruit, a cell, or a portion thereof. 8.An F₁ hybrid pepper plant having ‘E20S4216’ as a parent where ‘E20S4216’is grown from the seed of claim
 1. 9. Pollen or an ovule of the plant ofclaim
 2. 10. A protoplast produced from the plant of claim
 2. 11. Atissue culture produced from protoplasts or cells from the plant ofclaim 2, wherein said cells or protoplasts are produced from a plantpart selected from the group consisting of leaf, anther, pistil, stem,petiole, root, root tip, fruit, seed, flower, cotyledon, hypocotyl,embryo and meristematic cell.
 12. A pepper plant regenerated from thetissue culture of claim 11, wherein the plant has all of themorphological and physiological characteristics of the pepper plant ofclaim
 2. 13. A method of making pepper seeds, said method comprisingcrossing the plant of claim 2 with another pepper plant and harvestingseed therefrom.
 14. A method of making hybrid pepper ‘E20S4216’, saidmethod comprising selecting seeds from the cross of one ‘E20S4216’ plantwith another ‘E20S4216’ plant, a sample of ‘E20S4216’ pepper seed havingbeen deposited under ATCC Accession Number X1.